Corneal endothelium-like sheet and method of constructing the same

ABSTRACT

It is our intention to provide a transplantation material applicable to the treatment of various diseases that require corneal endothelium transplantation. Corneal endothelial cells are collected, proliferated and then sown on amnion and cultured. Thus, a corneal endothelium-like sheet, which has a cell layer made up of the corneal endothelium-origin cells formed on the amnion, can be obtained.

TECHNICAL FIELD

The present invention relates to corneal endothelium-like sheets andmethods of constructing the same. The corneal endothelium-like sheetprovided by the present invention can be used as a transplantationmaterial for treating diseases that require corneal endothelialtransplantation such as bullous keratopathy, corneal edema,keratoleukoma and keratoconus.

BACKGROUND ART

In recent years, the proliferation in the number of intraocularoperations has rapidly increased the number of postoperative diseases inwhich irreversible disorders occur in the corneal endothelium, resultingin corneal opacity. At the present time, the treatment used for suchdiseases in the corneal endothelium is penetrating keratoplasty.However, postoperative rejection of the endothelium followingpenetrating keratoplasty, is one of the largest problems in the clinicalfield of ophthalmology, and the development of a new technique to helpprevent rejection has eagerly been awaited.

Meanwhile, the normal anterior ocular segment is well known to be animmune privileged site concerned with anterior chamber associated immunedeviation. Factors constituting this immunosuppressive environment;immunosuppressive agents such as transforming growth factor beta(TGF-β), vasoactvie intestinal peptide (VIP), which are produced in thecornea and the iris, play an important role. Furthermore, it is reportedthat the production of immunosuppressive agents from the anterior ocularsegment is closely related to the corneal sensory nerve running throughthe corneal stroma and that production of the immunosuppressive agent issuppressed when the corneal sensory nerve is cut during penetratingkeratoplasty (Streilein J W, Bradley D, Sano Y, Sonoda Y:Immunosuppressive properties of tissues obtained from eyes withexperimentally manipulated corneas. Invest Ophthalmol Vis Sci.37(2):413-424, 1996. Sano Y, Streilein J W: Effects of corneal surgicalwounds on ocular immune privilege. In: Nussenblatt R B, Whitcup S M,Caspi R R, and Gery I eds. Advances in Ocular immunology. Amsterdam,ELSEVIER:207-210, 1994.).

Therefore, in those corneal endothelial diseases for which penetratingkeratoplasty is performed in order to minimize the failure of theimmunosuppressive environment of the anterior ocular segment, if onlydamaged endothelial cells can be replaced by healthy endothelial cells,it becomes an ideal operating technique as one can expect rejection tobe suppressed. Also, since only the posterior surface of the cornea isreplaced, corneal astigmatism occurs less frequently after operation, sorecovery of visual acuity from an early stage is expected.

Since a substrate as a carrier is essential for the transplantation ofcorneal endothelial cells, it is necessary to produce a sheet in whichcorneal endothelial cells are layered on the substrate in vitro.Conventionally, it has been thought that a corneal endothelial cell doesnot have proliferation potency, but proliferation has been confirmed invitro (Hyldahl L: Primary cell cultures from human embryonic corneas. JCell Sci. 66: 343-351, 1984. Senoo T, Obara Y, Joyce N: EDTA: A promoterof proliferation in human corneal endothelium. Invest Ophthalmol VisSci. 41: 2930-2935, 2000. Miyata K, Drake J, Osakabe Y, et al.: Effectof donor age on morphologic variation of cultured human cornealendothelial cells. Cornea. 20: 59-63, 2001).

Therefore, it is thought that if an appropriate substrate and culturecondition are found, a corneal endothelial cell sheet can be produced.On the other hand, it has been suggested that stem cells are present inthe peripheral portion of the corneal endothelial cell layer (Bednarz J,Engelmann K: Indication for precursor cells in the adult human cornealendothelium. Invest Ophthalmol Vis Sci. 42(suppl): S274, 2001). So, ifcorneal endothelium stem cells with high proliferation potency can becultured and proliferated in vitro, from a small number of cornealendothelial cells, it may be possible to produce a corneal endothelialsheet which can be used as a transplantation material. In particular, ifa residual autologous corneal endothelial cell is used, a rejection-freecorneal transplantation can probably be developed.

The present invention has been conceived under the above-mentionedcircumstances. The aim of this invention is to provide a cornealendothelium-like sheet that can be used for corneal endothelialtransplantation to treat corneal endothelial diseases.

DISCLOSURE OF INVENTION

The present inventors have investigated earnestly in view of theabove-mentioned conditions. Specifically, they have investigated theselection of a substrate suitable for culturing corneal endothelialcells in vitro and a method for forming cell layers with a high celldensity. Firstly, corneal endothelial cells were collected, and thencultured and proliferated in vitro. A cell suspension with high celldensity was produced by subculturing the proliferated cells andsubjecting them to appropriate centrifugation. Then, as a substrate(carrier), amniotic membrane containing collagen as a main component wasemployed, and the cell suspension was planted thereon and cultured for apredetermined time. As a result, a single layered cell layer, in whichcells derived from the corneal endothelial cells have a similarmorphology to that of the living body, was formed. When this cell layerwas observed, it had the equivalent cell density to the cornealendothelial cells of a living body and had a configuration in whichhexagonal shaped cells were regularly layered to form a single layerstructure. On the other hand, when the resultant composition sheetconsisting of amniotic membrane and a cell layer was transplanted in arabbit from which a part of the corneal endothelial cell layer had beenremoved, good survival was observed. It was confirmed that thecomposition could be suitably used as a material for corneal endothelialcell transplantation. The present invention was completed based on theabove-mentioned findings and provides the following configurations.

[1] A corneal endothelium-like sheet comprising: a collagen layer and acell layer formed on the collagen layer; the cell layer consisting ofcells derived from corneal endothelium.

[2] A corneal endothelium-like sheet described in [1], wherein thecollagen layer is derived from amniotic membrane.

[3] A corneal endothelium-like sheet described in [1], wherein thecollagen layer consists of amniotic membrane from which the epitheliumhas been removed.

[4] A corneal endothelium-like sheet comprising a cell layer consistingof cells derived from corneal endothelium.

[5] A corneal endothelium-like sheet described in any of [1] to [4],wherein the cell layer has a monolayer structure.

[6] A corneal endothelium-like sheet described in any of [1] to [5],wherein the cell density of the cell layer is about 2000 cells/mm² toabout 4000 cells/mm².

[7] A corneal endothelium-like sheet described in any of [1] to [6],wherein the plane view shape of the cells derived from cornealendothelium is substantially hexagonal.

[8] A corneal endothelium-like sheet described in any of [1] to [7],wherein the cells derived from corneal endothelium are arrangedregularly in the cell layer.

[9] A method for constructing a corneal endothelium-like sheet, themethod comprising the following steps:

-   -   a) culturing and proliferating collected corneal endothelial        cells;    -   b) collecting the proliferated corneal endothelial cells and        producing a cell suspension;    -   c) planting the cell suspension on a collagen layer and        culturing thereof.

[10] A method for producing a corneal endothelium-like sheet describedin [9], wherein the following step is carried out after step b):

-   -   b-1) increasing the cell density in the cell suspension using        centrifugation.

[11] A method for producing a corneal endothelium-like sheet describedin [9] or [10], wherein centrifugation is carried out after planting thecell suspension in step c).

[12] A method for producing a corneal endothelium-like sheet describedin [9] or [10], wherein step c) comprises the following procedures:

-   -   c-1) placing a container in a culture container, the container        having a bottom surface consisting of a membrane with a pore        size capable of allowing a culture solution to pass through;    -   c-2) forming acollagen layer on the bottom face of the        container;    -   c-3) planting the cell suspension on the collagen layer;    -   c-4) carrying out centrifugation;    -   c-5) culturing.

[13] A method for producing a corneal endothelium-like sheet describedin any of [9] to [12], wherein the collagen layer is derived fromamniotic membrane.

[14] A method for producing a corneal endothelium-like sheet describedin any of [9] to [12], wherein the collagen layer consists of amnioticmembrane from which the epithelium has been removed.

Note that the “corneal epithelium-like sheet” herein is used as a termfor meaning a composition that has a similar feature to a cornealendothelial cell layer and can be transplanted in order to reconstruct acorneal endothelial cell layer. Furthermore, unless otherwise specified,“corneal endothelial cell” is used as a term encompassing cells includedin a corneal endothelial cell layer. That is to say, it also includescorneal endothelial stem cells.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the morphology of a corneal endothelium like cell layerformed on amniotic membrane from which the epithelium has been removed.A shows an HE (hematoxylin-eosin) stained image; B shows an alizarinstained image; and C and Dare scanning electron microscope images.

FIG. 2 shows slit photographs of an anterior ocular segment at day 4after transplantation via full thickness trepanation. A shows a controlgroup (a group in which the Descemet's membrane has been removed); Bshows a group with amniotic membrane (AM group); and C shows a groupwith a corneal endothelium-like sheet.

FIG. 3 shows slit photographs of an anterior ocular segment at day 7after transplantation via full thickness trepanation. A shows a controlgroup (a group in which the Descemet membrane has been removed); B showsan AM group; and C shows a group using a corneal endothelium-like sheet.

FIG. 4 is a graph showing the changes in corneal thickness before andafter transplantation via full thickness trepanation. In this graph, *represents p<0.05. In the group where a corneal endothelium-like sheetwas used, the corneal thickness is reduced significantly (p<0.05) ascompared with the control group and the AM group. FIG. 5 is across-sectional view schematically showing the state of the instrumentswhen endothelial cells are cultured. In a culture dish (1), a cultureinsert (2) is disposed. Furthermore, it shows the state in which thecorneal endothelial cells (3) are cultured on the bottom face of theculture insert (2). Reference numeral 5 denotes a culture medium.

FIG. 6 is a cross-sectional view schematically showing the state of theinstruments when endothelial cells are cultured on amniotic membrane. Ina culture dish (1), a culture insert (2) is disposed. It also shows thesituation in which amniotic membrane (4) is extended on the bottom faceof the culture insert (2) and cells (3) derived from the cornealendothelial cells are cultured thereon. Reference numeral 5 denotes aculture medium.

1: culture dish, 2: culture insert (container for inserting culture), 3:cells derived from the corneal endothelial cell, 4: amniotic membrane,5: culture medium.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention relates to a corneal endothelium-like sheetcomprising a collagen layer, and a cell layer formed on the collagenlayer, the cell layer consisting of cells derived from cornealendothelium.

Herein, the types of collagen constituting the collagen layer are notlimited in particular, but it is preferable for the collagen layer to bederived from amniotic membrane. Furthermore, it is also preferable touse amniotic membrane from which the epithelium has been removed by suchmethods as scraping treatment. Whether or not the collagen layer is madeof amniotic membrane from which the epithelium has been removed can beconfirmed by examining to check that a cell from the amniotic membraneepithelium layer is not contained in the collagen layer. It ispreferable that human amniotic membrane is used. Note that “derived fromamniotic membrane” broadly means that amniotic membrane is used as astarting material.

On the other hand, “cells derived from corneal endothelium” means cellsproduced by culturing the collected corneal endothelial cells, so thatthey are proliferated and differentiated.

It is preferable for the corneal endothelial like sheet to have some, ormore suitably, all of the characteristics or features mentioned below.

(1) The cell layer has a monolayer structure (single-layered structure).This is one of the features of the corneal endothelial cell layer in theliving body.

(2) The cell density of the corneal endothelial cell layer of the livingbody is said to be about 4000 cells/mm² in a newborn human. It decreasesin accordance with growth and aging and reaches about 2000 cells/mm² toabout 3000 cells/mm² in adult humans in the normal state. In view ofthis fact, it is preferable for the cell density of the cell layerconstituting the corneal endothelium-like sheet presented in thisinvention, to be about 2000 cells/mm² to about 4000 cells/mm². Inparticular, in a case where a recipient (a person to be transplanted) isan adult, it is preferable for the cell density to be about 2000cells/mm² to about 3000 cells/mm².

(3) The plane view shape of cells constituting the cell layer ishexagonal. This is one of the features of the corneal endothelial cellsin the living body. When this feature is observed, it is expected thatthe corneal endothelium-like sheet should be similar to the cornealendothelium cell layer and should exhibit similar functions to those ofthe corneal endothelial cell layer.

(4) In the cell layer, cells are regularly arranged. It is thought thatin the corneal endothelial cell layer of the living body, cellsconstituting the corneal endothelial cell layer are regularly arranged,so that high transparency is maintained and the moisture controlfunction of the cornea is exhibited appropriately. Therefore, when sucha morphological feature is provided, the corneal endothelium-like sheetin this present invention should exhibit similar functions to those ofthe corneal endothelial cell layer in the living body.

The corneal endothelium-like sheet in this present invention can beproduced, for example, by the following method.

<1> The Collection and Proliferation of Corneal Endothelial Cells.

Corneal endothelial cells are collected from the cornea of the recipienthim/herself or from an appropriate donor, in the usual manner. Forexample, the Descemet's membrane and the endothelial cell layer of thecorneal tissue are peeled off from the corneal stroma, then transferredto a culture dish and treated with dispase. Thus, corneal endothelialcells are peeled away from the Descemet's membrane. The cornealendothelial cells residing in the Descemet's membrane can be peeled of fby such means as pipetting. The Descemet's membrane is removed, afterwhich, corneal endothelial cells are cultured in an appropriate culturesolution. For a culture solution, it is possible to use, for example, acommercially available DMEM (Dullbecco's Modified Eagle's Medium) towhich FBS (fetal bovine serum), b-FGF (basic-fibroblast growth factor),EGF (epidermal growth factor), insulin and antibiotics such aspenicillin and streptomycin are added. It is preferable to use a culturecontainer (culture dish) the surface of which is coated with type Icollagen, type IV collagen, fibronectin, laminin or extracellular matrixof bovine endothelial cells. This is advantageous because the attachmentof the corneal endothelial cells to the surface of the culture containeris promoted, thus enabling excellent proliferation.

The temperature conditions for culturing corneal endothelial cells arenot necessarily limited as long as the corneal endothelial cells cangrow. However, the temperature should be for example, about 25° C. toabout 45° C. When considering proliferation efficiency, a preferabletemperature is about 30° C. to 40° C.; more specifically, 37° C. Theculture time varies depending upon the state of cells to be used and soon but generally, it is 7 to 14 days.

It is preferable to use corneal endothelial cells from the recipienthim/herself, if possible. This is advantageous because a cornealendothelium-like sheet, that may be free from immunorejection when it isemployed for transplantation, can be produced. Thus transplantation thatis not accompanied by immunorejection can be performed. When it isimpossible or difficult to obtain corneal endothelial cells from therecipient him/herself, corneal endothelial cells from a person otherthan the recipient may be used. However, in this case, it is preferableto select a donor by considering immunocompatibility.

<2> Subculture and Production of Cell Suspension.

After the corneal endothelial cells used for culture are proliferated, asubculture of the cells can be carried out. Preferably, a subculture iscarried out when cells become subconfluent or confluent. The subculturecan be carried out as follows. Firstly, cells are peeled off from thesurface of the culture container by treating with trypsin-EDTA. Then,cells are collected. Culture solution is added to the collected cells tocreate a cell suspension. It is preferable for centrifugation to becarried out when or after cells have been collected. With suchcentrifugation, cell suspension with a high cell density can beprepared. Note here that conditions for centrifugation can include 500rpm (30 g) to 1000 rpm (70 g) and 1 to 10 minutes.

As in the above-mentioned initial culture, a cell suspension is plantedon the culture container and cultured. The subculture can be carried outin the same culture conditions as the above-mentioned initial culture.The culture time is different depending upon the state of the cells tobe used, but generally, it is 7 to 21 days. The above-mentionedsubculture can be carried out a number of times if necessary. Byrepeating the subcultures, the number of cells can be increased and acell suspension with a high cell density can be prepared. Finally, it ispreferable to prepare a cell suspension with a cell density of about5×10⁵ cells/ml to 20×10⁵ cells/ml.

<3> Planting of the Cell Suspension and Culture

A cell suspension is planted on a collagen layer and cultured. At thistime, the number of cells is adjusted so that a cell layer with adesired cell density can be formed in the end product; the cornealendothelium-like sheet. About 3000 cells/mm² to 7500 cells/mm²,(preferably about 5000 cells/mm² to 7500 cells/mm²) are planted so thata cell layer with a cell density of about 2000 cells/mm² to 4000cells/mm² is formed. The culture is carried out under the sameconditions as in the above-mentioned initial culture. The culture timevaries depending on the state of cells to be used, but generally it is,for example, 3 to 21 days.

There are no limtitations as to the type of collagen used as a materialfor the collagen layer; type I, type III, type IV and type VIIIcollagens can be used. Also, a number of collagens can be used incombination. Such collagens can be extracted and purified, using an acidsolubilization, alkali solubilization, and oxygen solubilization method,from the connective tissue of such areas as the skin and cartilage ofanimals like swine, bovine, sheep, etc. Note that for the purpose oflowering antigenicity, a so-called atherocollagen should be obtained byremoving telopeptide via treatment using a catabolic enzyme such aspepsin or trypsin.

It is preferable that, for the collagen layer, a collagen derived fromamniotic membrane, from human amniotic membrane in particular, is used.Herein, the collagen layer “derived from amniotic membrane” broadlymeans that the collagen layer is obtained using amniotic membrane as astarting material. Human amniotic membrane is a membrane covering theoutermost layer of the uterus and the placenta, and a basal membrane andan epithelium layer are formed on parenchymal tissue that is rich incollagen. Human amniotic membrane can be collected from human embryonicmembrane or placenta at the time of delivery. Specifically, humanamniotic membrane can be prepared by treating and purifying theintegrated material including human embryonic membrane, placenta, andumbilical cord obtained right after delivery. The method of treating andpurifying can be employed using the method described in JP 5(1993)-56987A. That is to say, amniotic membrane is detached from theembryonic membrane at time of delivery and the remaining tissue isremoved by physical treatment such as ultrasonic cleansing and an enzymetreatment. Then, an appropriate cleaning process is carried out and thusthe human amniotic membrane can be prepared.

The prepared human amniotic membrane can be cryopreserved before use.The human amniotic membrane can be frozen, for example, in a liquidmixing equal volume ratio of DMEM (Dulbecco's modified Eagle's medium)and glycerol at −80° C. Using cryopreservation, not only an improvementin the operation but also a reduction in antigenicity can be expected.

Intact amniotic membrane may be used as a collagen layer but it ispreferable for amniotic membrane, from which the epithelium has beenremoved by such methods as scraping treatment, to be used. For example,after thawing, cryopreserved human amniotic membrane is subjected totreatment with EDTA or proteolytic enzyme so as to loosen adhesionbetween the cells and then the epithelium is scraped by using a cellscraper. Thus, the human amniotic membrane from which the epithelium hasbeen removed, can be prepared.

When human amniotic membrane, from which the epithelium has beenremoved, is used as a collagen layer, it is preferable that cornealendothelial cells are planted at the side of the surface from which theepithelium has been removed and exposed to the surface (i.e., the sideof the basement membrane). This is advantageous because it is thoughtthat the side of this surface contains a large amount of type IVcollagens and the planted corneal endothelial cells can adhere andproliferate well.

Culture of the corneal endothelial cells on the collagen layer can becarried out, for example, by the following procedures. Firstly, acontainer with a bottom face made of membrane with a suitable pore sizethrough which culture solution can pass (hereinafter, also referred toas a “culture insert”) is placed with the bottom face faced downward.Then, a collagen layer is formed on the bottom face of the cultureinsert (inner side of the culture insert). Then, on the collagen layer,the cell suspension is planted and cultured. Note here that the collagenlayer may be formed on the bottom face of the culture insert in advance.That is to say, for example, a culture may be carried out by disposingamniotic membrane from which the epithelium has been removed on thebottom face of the culture insert (drying treatment may be carried outonce in this state), then setting this culture insert in a culturecontainer, and finally planting and culturing the cell suspension.

An example of a membrane that can be used for the bottom face of theculture insert includes a polycarbonate membrane, having a pore size ofabout 0.4 μm to 3.0 μm. Besides this, a polyester membrane may be used.Such membranes are widely commercially available.

Centrifugation may be carried out after the cell suspension is plantedin the culture insert. Thereby, the cell density on the collagen layercan be increased. Furthermore, adhesion of cells to the surface of thecollagen layer becomes excellent. Furthermore, an effect of reducing thedeviation of cell density can be obtained. Note here that conditions ofcentrifugation herein include, for example, 500 rpm (30 g) to 1,000 rpm(70 g) and 1 to 10 minutes.

By culturing, as mentioned above, a corneal endothelium like sheet, inwhich a cell layer made of cells derived from corneal endothelium isformed on a collagen layer, can be formed. The corneal endothelium-likesheet can be used as a transplantation material (substitute for thecorneal endothelium) for treating diseases that require transplantationof the corneal endothelium, such as bullous keratopathy, corneal edema,keratoleukemia and keratoconus. Note here that a sheet obtained byremoving part or all of the collagen layer (that is, only the celllayer) may be used as a graft. The collagen layer can be removed byappropriately combining a chemical treatment with EDTA, an enzymatictreatment using proteolytic enzyme, and physical treatment such asscraping using forceps.

Culture (including initial culture, subculture and culture on thecollagen layer) of corneal endothelial cells can be carried out in thecoexistence of supporting cells. The supporting cell is also referred toas a feeder cell and supplies the culture solution with a growth factor.When the corneal endothelial cells are cultured in the coexistence ofsupporting cells, improvement in the proliferation efficiency and thepromotion corneal endothelial cell differentiation can be expected. Withregard to the supporting cells, the likes of 10T1/2 fibroblast (HyldahlL: Primary cell cultures from human embryonic corneas. J Cell Sci. 66:343-351, 1984), a 3T3 cell (Swiss mouse 3T3 cell, mouse NIH3T3 cell,3T3J2 cell or a corneal stromal cell may be used.

When the corneal endothelial cells are cultured in the coexistence ofsupporting cells, it is preferable for an isolation membrane, with apore size through which the supporter cells cannot pass, to be provided.This should be between the corneal endothelial cells and the supportingcells. The use of this isolation membrane makes it possible to preventthe supporting cells from entering the side of the corneal endothelialcells at the time of culture. As a result, the supporting cells shouldnot be mixed in the final product; the corneal endothelium-like sheet.This means that a corneal endothelium-like sheet free from immunologicalrejection problems via the supporting cells, can be constructed. This isextremely clinically significant.

When supporting cells are used, it is preferable for them to beinactivated with mitomycin C. This is advantageous because theinhibition of the proliferation of the corneal endothelial cells, due tothe proliferation of the supporting cells themselves, is prevented. Suchinactivation may be carried out using radiation treatment.

As for the isolation membrane, a membrane similar to that used in theabove-mentioned culture insert can be employed. For example, a membranewith a pore size of about 0.4 μm to 3.0 μm made of polycarbonate orpolyester, can be used.

The cell density of the supporting cells may be, for example, about1×10² cells/cm² or more, preferably in a range from about 1×10²cells/cm² to 1×10⁷ cells/cm², and even more preferably, in a range fromabout 1×10³ cells/cm² to 1×10⁵ cells/cm². It is not good for the numberof supporting cells to be small, because it is thought that theproliferation rate of the corneal endothelial cells is lowered. On theother hand, the number of supporting cells shouldn't be too large,because then the proliferation rate of the corneal endothelial cells issomewhat lowered.

Transplantation methods can include full thickness trepanation and deepkeratectomy. In the former method, full thickness cornea is obtainedusing a trephine and the corneal endothelial cell layer is replaced bythe corneal endothelium-like sheet, resulting in the return to a fullthickness cornea for the recipient. Specifically, the method can becarried out as follows. Firstly, incision of the full thickness corneaof a recipient (host) is carried out by using a trephine, and a part (orthe whole) of the cornea is collected as a button shape. Then, from thepiece of cornea collected, the Descemet's membrane and a cornealendothelial cell layer are peeled off. The corneal endothelium-likesheet is then stuck onto the exposed corneal stroma. At this time, thecorneal endothelium-like sheet is adhered so that the collagen layerfaces the side of the corneal stroma. Thereafter, the corneal graft isreturned to the recipient and fixed with a suture.

On the other hand, in the case of deep keratectomy, instead ofextracting the full thickness of the cornea, only the deep portion ofthe cornea is excised. This method is thought to have a reduced burdenon behalf of the recipient. Specifically, the method can be carried outas follows. Firstly, a part of the recipient's corneal stroma isdelaminated, and the posterior part of corneal stroma and the Descemet'smembrane or the endothelial cell layer are excised. Note here that onlythe endothelial cell layer or only the endothelial cell layer andDescemet's membrane may be peeled off and excised. Next, the cornealendothelium-like sheet is inserted into the excised portion by using aspatula. If necessary, air is pumped into the anterior chamber and thegraft is fixed. Furthermore, in the case of a corneal endothelium layerwith no collagen layer, only the recipient's affected cornealendothelial cells should be peeled away.

Note that here, it can be confirmed whether or not the transplantedcorneal endothelium-like sheet has a barrier function; a pump functioncan be exhibited, similar to the corneal endothelial cell layer in theliving body, by examining the change in corneal thickness aftertransplantation and the occurrence of edema.

EXAMPLE 1 Collection of Amniotic Membrane

Amniotic membrane was obtained during Caesarean section from theplacenta of a pregnant woman, who did not have any systemiccomplications. Sufficient, informed consent was obtained from both thepatient and the obstetrician in advance. The operation was carried outcleanly. In accordance with the operation guidelines, the operatorswashed their hands, and then wore special gowns. Before delivery, aclean vat for obtaining amniotic membrane and physiologic saline forwashing were prepared. After delivery, the placenta was transferred tothe vat and amniotic membrane tissue was manually removed from theplacenta. A portion where amniotic membrane and placenta were stronglyadhered to each other, was separated with scissors.

EXAMPLE 2 Treatment of Amniotic Membrane

The treatment process of amniotic membrane included: (1) washing, (2)trimming, and (3) storing sequentially, in this order. Throughout theseprocesses, the operation should be carried out in a clean draft.Sterilized containers and instruments were used and in the case ofdishes, sterilized disposable ones were used. The amniotic membraneobtained, was washed to remove blood components and further washed in asufficient amount of physiological saline (0.005% ofloxacin was added).Then, the amniotic membrane was transferred to a phosphate buffersolution (PBS) in a dish and cut and divided into a size of about 4×3 cmwith scissors. The divided pieces of amniotic membrane were stored inseveral dishes filled with a stock solution, and thereafter thoseamniotic membranes in good condition were selected.

EXAMPLE 3 Storage of Amniotic Membrane

1 cc each of stock solution was placed in 2 cc sterilized cryotube andone sheet of amniotic membrane, which had been obtained, washed andselected, was placed and labeled, then stored in a refrigerator at −80°C. For the stock solution, 50% sterilized glycerol in DMEM (Dulbecco'sModified Eagle Medium: GIBCOBRL) was used. The expiration date for useof stored amniotic membrane was determined at 3 months and expiredamniotic membrane was disposed of by incineration.

EXAMPLE 4 Treatment of Amniotic Epithelium

Amniotic membrane was subjected to treatment to remove the epitheliumand was then used for culture. Firstly, amniotic membrane stored at −80°C. was thawed at room temperature, and then washed thoroughly in a dishof sterilized phosphate buffer solution (PBS). After washing, theamniotic membrane was stored in a 0.02% EDTA solution (Nacalai tesque)at 37° C. for 2 hours, and then the epithelium was mechanically scrapedout by using a cell scraper (Nunc, USA) and used as a substrate forculture. Note that it was confirmed that one layer of the amnioticepithelium was completely scraped using this procedure by opticalmicroscope and electron microscope (scanning electron microscope)operations.

EXAMPLE 5 Collection and Initial Culture of Human Corneal EndothelialCells

The Descemet's membrane and an endothelial cell layer were peeled offfrom the stroma of human (age: 47, male) corneal tissue by using aforceps, then disposed on a 35 mm culture dish, and treated with 1 ml ofdispase (final concentration: 1.2 U/ml) that had been double dilutedwith PBS containing no calcium under conditions of 37° C. and in 5% CO₂for 60 minutes. With this treatment, many of the corneal endothelialcells dropped off from the Descemet's membrane. In order to aid thedropping off of the corneal endothelial cells residing in the Descemet'smembrane, pipetting treatment was carried out. Next, the Descemet'smembrane was removed and the corneal endothelial cells were transferredto a 15 cc centrifugation tube. A culture solution was added to thecentrifugation tube to make a total amount of 5 ml and thencentrifugation was carried out under conditions of 1000 rpm, 70 g for 3minutes. For the culture solution, DMEM (GIBCOBRL) was used, to which10% FBS (Dainippon Pharmaceutical Co., Ltd), 2 ng/ml b-FGF (Invitrogen),penicillin (50 U/ml)-streptomycin (50 μg/ml) (Nacalai tesque) wereadded. Supernatant was removed and then culture solution was added toform about 1 ml of cell suspension. This cell suspension was planted ona 24-hole culture dish coated with type IV collagen and cultured underconditions of 37° C. and in 5% CO₂. Thereafter, the culture solutionswere exchanged for new solution every 48 hours.

EXAMPLE 6 Subculture of Endothelial Cells

When the cultured cells became confluent, the culture solution wasremoved and the cells were washed three times with 1 ml of PBScontaining no calcium. After washing, 200 μl of 0.05% trypsin-EDTA wasadded and placed under conditions of 37° C. and 5% CO₂ for 3 minutes.With this treatment, endothelial cells, adhered to the bottom surface ofthe culture dish, were allowed to float. 5 ml of cultured solutions wereadded and the cell suspension was transferred to a 15 cc centrifugationtube and centrifugation was carried out under conditions of 1000 rpm, 70g for 3 minutes. Supernatant was removed and then culture solution wasadded to form about 2 ml of cell suspension. This cell suspension wasplanted on a 35 mm culture dish coated with type IV collagen andcultured under conditions of 37° C. and 5% CO₂. Thereafter, the culturesolutions were exchanged for new solutions every 48 hours. When thecells became confluent, the same treatment as mentioned above wascarried out, and the subculture was repeated until the necessary numberof cells were obtained. When the subculture was repeated 5 times, thecell density was measured. The cell density was 500 to 1000 cells/mm².Note that by carrying out a subculture using the above-mentionedtreatments, at least 10 generations of subculture were achievable.

EXAMPLE 7 Production of High Density Cultured Corneal Endothelial Cells

The subcultured cells were treated with 0.05% trypsin-EDTA and then thenumber of cells was counted. Subsequently, centrifugation was carriedout and supernatant was removed. Then, a culture solution was added sothat the cell density became 2.0×10⁶ cells/ml. By using the obtainedcell suspension, a corneal endothelial cell layer was produced via thefollowing procedures. As a culture instrument, a 6-hole culture dish(Corning, N.Y.) and a culture insert (culture insert: culture insertingcontainer; made of polycarbonate; average pore size: 3.0 μm; produced byCorning, N.Y.) were used. For a culture solution, the same culturesolution mentioned above was used.

Firstly, a culture insert was disposed in a culture dish. Then, cellsuspension was planted in the culture insert so that the cell densityreached about 5000 cells/mm². Thereafter, the culture dish wascentrifuged under conditions of 1000 rpm, 70 g for 3 minutes. Aftercentrifugation, culture was carried out under conditions of 37° C. and5% CO₂. As a result, after culture for 14 days, a cell layer with a celldensity of about 3000 cells/mm² was obtained. Furthermore, in a 2-weekculture, no morphological changes in cells were observed.

Note here that FIG. 5 is a cross-sectional view schematically showingthe state during culturing. It shows a state in which the culture insert(2) is disposed in a culture dish (1), and the corneal endothelial cells(3) are cultured in the culture insert 2. Reference numeral 5 denotes aculture medium.

EXAMPLE 8 Production of a Corneal Endothelium-Like Sheet Using AmnioticMembrane as a Substrate

By using a cell suspension (cell density: about 2.0×10⁶ cells/ml), whichis also used in Example 7, a corneal endothelium-like sheet was producedvia the following procedures. For culture instruments, the sameinstruments as in Example 7 were used.

Firstly, a culture insert was disposed in a culture dish. Then, amnioticmembrane from which the epithelium had been scraped out, obtained inExample 4, was extended and laid with the side from which the epitheliumhad been scraped out facing upward. Subsequently, cell suspension wasplanted on the amniotic membrane so that the cell density reached about5000 cells/mm². Thereafter, the culture dish was centrifuged underconditions of 1000 rpm, 70 g for 3 minutes. After centrifugation,culture was carried out under conditions of 37° C. and 5% CO₂ for about14 days. As a result, as in the living body, a continuoussingle-structured cell layer was constructed (FIG. 1A). When the celldensity of the cell layer was measured, it was 3340 cells/mm², whichshowed a similar cell density to that of the cell layer obtained inExample 7 (FIG. 1B). In addition, when the morphology was observed byusing a scanning electron microscope, cells constituting the cell layerhad a hexagonal, flat shape, similar to the endothelial cells of theliving body (FIGS. 1C and 1D). Furthermore, it was observed that a celllayer was configured in which these hexagonal-shaped cells were arrangedmore or less uniformly.

Note that FIG. 6 is a cross-sectional view schematically showing theconditions during culturing. In a culture dish (1), a culture insert (2)is disposed. Furthermore, it shows that on the bottom face of theculture insert (2), amniotic membrane (4) is extended and the cornealendothelial cells (3) are cultured thereon. Reference numeral 5 denotesa culture medium.

EXAMPLE 9 Transplantation of the Corneal Endothelium-Like Sheet

a. Transplantation by Full Thickness Trepanation.

Full thickness of the central portion of the cornea (host cornea) in arabbit (aged 6-weeks, Japanese white color species) was incised with atrephine of 7 to 8 mm in diameter. The Descemet's membrane of the hostcorneal graft obtained in a button shape was peeled off, including theendothelial cell layer. The corneal endothelium-like sheet obtained inExample 8 was taken out from the culture dish with a forceps, followedby incision with a 6 to 8 mm trephine and placed on the stroma of thehost cornea from which the Descemet's membrane and endothelial cell hadbeen removed. At this time, a suture was not used and the cornealendothelium-like sheet was adhered to the corneal stroma by absorbingwater with a sponge and drying slightly. The host corneal graft in whichthe corneal endothelium was disposed on amniotic membrane was sutured tothe host cornea with 10-0 nylon thread. Thereafter, every day until day7 after transplantation, the thickness of the transplanted portion ofthe cornea and the state of the edema were observed. Note that as acontrol for comparison, a group (AM group) using amniotic membraneinstead of the corneal endothelium-like sheet and a group (controlgroup), to which a host corneal graft, from which the Descemet'smembrane with endothelial cells had been removed, were used.

FIG. 2 shows the state of the transplanted site at day 4 aftertransplantation. In the group in which the Descemet's membrane wasremoved, and transplantation was carried out (control group),significant edema was observed (FIG. 2A). Furthermore, also in the AMgroup, as in the control group, significant edema was observed (FIG.2B). On the other hand, in the cornea to which the cornealendothelium-like sheet was transplanted, compared with the control andAM groups, edema was significantly reduced and transparency remained. Atthe time of observation, day 7 after transplantation, in the cornea towhich the corneal endothelium-like sheet was transplanted, as comparedwith the control group (FIG. 3A) and the AM group (FIG. 3B), edema wasreduced and transparency was maintained (FIG. 3C). In addition, in thecornea to which the corneal endothelium-like sheet was transplanted, thecorneal thickness was significantly reduced as compared with the controland AM groups (FIG. 4).

The above mentioned results suggest that, on amniotic membrane, cellsderived from the human corneal endothelial cells had a morphology thatwas extremely similar to that of the cells in a living body.Furthermore, by using centrifugation, it was possible to obtain a celldensity of about 3000 cells/mm². It was also shown that the cornealendothelium-like sheet produced by this technique could perform itsfunction in vivo.

b. Transplantation by Deep Keratectomy

The corneal (host cornea) stroma of a rabbit (aged 6-weeks, Japanesewhite color species) was peeled away and a deep layer of the parenchymastroma, with a diameter of 7 to 8 mm in the central portion, wasincised, including the endothelial cell layer. Alternately, only theendothelial cells and the Descemet's membrane, with a 7 to 8 mm diameterin the central part, were peeled off and removed from the anteriorchamber. The corneal endothelium-like sheet obtained in Example 8 wasinserted into the excised portion using a spatula, from between thestromal layers or into the anterior chamber. Then, by pumping air intothe anterior chamber, the graft was fixed. Thereafter, every day untilday 7 after transplantation, the thickness of the transplanted portionof the cornea and the state of edema were observed. As a result, in thecornea to which the corneal endothelium-like sheet was transplanted, asin full thickness trepanation, the corneal thickness and corneal edemawere significantly reduced compared to the AM group to which onlyamniotic membrane was transplanted or the control group in which onlyremoval of the corneal endothelium was carried out.

From the examples mentioned above, it was confirmed that the cornealendothelium-like sheet performed its function in vivo. That is to say,it has been confirmed that the corneal endothelium-like sheet produced,functions excellently as a substitute for corneal endothelium and can beused as a transplantation material for reconstructing the cornealendothelium when the corneal endothelium is damaged or affected.Furthermore, it has been proven that by using this sheet, an idealoperating technique capable of minimizing the failure of theimmunosuppressive environment in the anterior ocular segment andreplacing only the damaged corneal endothelial cells with healthyendothelial cells, can be achieved.

The present invention is not limited to the description of the aboveembodiments. A variety of modifications, which are within the scopes ofthe following claims and which are easily achieved by a person skilledin the art, are included in the present invention.

INDUSTRIAL APPLICABILITY

This present invention; the corneal endothelium-like sheet, has astructure similar to the corneal endothelial cell layer of the livingbody and can be used as a transplantation material for treating variousdiseases that require corneal endothelial cell transplantation. Thecorneal endothelium-like sheet has excellent survival propertiesfollowing transplantation, performs the functions of the cornealendothelial cell layer, i.e., a barrier function and a pump function,extremely useful in the reconstruction of the damaged cornealendothelial cell layer. Meanwhile, this corneal endothelium-like sheetis formed by culturing and proliferating the collected corneaendothelial cells in vitro. Therefore, it is possible to produce atransplantation material based on a small amount of corneal endothelialcells. Also in a patient with a reduced number of corneal endothelialcells, transplantation materials can be produced from auto-cornealendothelial cells. This means that corneal transplantation that does notcause rejection can be realized. Furthermore, a corneal endotheliumtransplantation technique in which only the damaged site is replaced,can be realized. An ideal operating technique that does not causecomplications during and after transplantation, which was a problem inconventional penetrating keratoplasty, can now be made available.

1. A corneal endothelium-like sheet, comprising: a collagen layer; and acell layer formed on the collagen layer, the cell layer consisting ofcells derived from corneal endothelium.
 2. A corneal endothelium-likesheet according to claim 1, wherein the collagen layer is derived fromamniotic membrane.
 3. A corneal endothelium-like sheet according toclaim 1, wherein the collagen layer consists of amniotic membrane fromwhich the epithelium has been removed.
 4. A corneal endothelium-likesheet comprising a cell layer consisting of cells derived from cornealendothelium.
 5. A corneal endothelium-like sheet according to claim 1,wherein the cell layer has a monolayer structure.
 6. A cornealendothelium-like sheet according to claim 1, wherein the cell density ofthe cell layer is about 2000 cells/mm² to about 4000 cells/mm².
 7. Acorneal endothelium-like sheet according to claim 1, wherein the planeview shape of the cells derived from corneal endothelium is hexagonal.8. A corneal endothelium-like sheet according to claim 1, wherein thecells derived from corneal endothelium are arranged regularly in thecell layer.
 9. A method for constructing a corneal endothelium-likesheet, the method comprising the following steps: a) culturing andproliferating collected corneal endothelial cells; b) collecting theproliferated corneal endothelial cells and producing a cell suspension;and c) planting the cell suspension on a collagen layer and culturingthereof.
 10. A method for producing a corneal endothelium-like sheetaccording to claim 9, wherein the following step is carried out afterstep b): b-1) increasing the cell density in the cell suspension usingcentrifugation.
 11. A method for producing a corneal endothelium-likesheet according to claim 9, wherein centrifugation is carried out afterplanting the cell suspension in step c).
 12. A method for producing acorneal endothelium-like sheet according to claim 9, wherein step c)comprises the following steps: c-1) placing a container in a culturecontainer, the container having a bottom surface consisting of amembrane with a pore size capable of allowing a culture solution to passthrough; c-2) forming a collagen layer on the bottom face of thecontainer; c-3) planting the cell suspension on the collagen layer; c-4)carrying out centrifugation; c-5) culturing.
 13. A method for producinga corneal endothelium-like sheet according to claim 9, wherein thecollagen layer is derived from amniotic membrane.
 14. A method forproducing a corneal endothelium-like sheet according to claim 9, whereinthe collagen layer consists of amniotic membrane from which theepithelium has been removed.